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Sperm motility is vital to human reproduction, and malformed sperm flagella can cause male infertility. Individuals with multiple morphological abnormalities of the flagella mostly have absent, short, coiled, bent, and/or irregular-caliber flagella. In this study, a patient with male infertility underwent a physical examination along with his wife. Genetic testing was performed by whole-exome sequencing of the couple, and Sanger sequencing was performed for validation. Novel biallelic variations in the DNAH1: (NM_015512.4) gene consisting of c.1336G>C (p.E446Q) and c.2912G>A (p.R971H) were identified. In silico structural analysis revealed that the amino acid residues affected by the variation were evolutionarily conserved, and the variant p.R971H influenced the stability of the DNAH1 protein. Morphological studies of the patient's sperm showed defects in its flagella. Results of Papanicolaou staining and scanning electron microscopy demonstrated coiled and short flagella with multiple anomalies. Transmission electron microscopy of the sperm flagella showed that the inner dynein arm and radial spoke were absent, and the dense fiber and microtubule doublets were displaced. Quantitative PCR of the mRNA of the patient's sperm showed that the expression of DNALI1 was dramatically reduced. Collectively, these findings elucidated the genetic cause of the family's infertility and provided insight into the functioning of the DNAH1 gene.

The classic mode of G protein‐coupled receptor (GPCR)‐mediated transactivation of the receptor tyrosine kinase epidermal growth factor receptor (EGFR) transactivation occurs via matrix metalloprotease (MMP)‐mediated cleavage of plasma membrane‐anchored EGFR ligands. Herein, we show that the Gαs‐activating GPCR ligands vasoactive intestinal peptide (VIP) and prostaglandin E2 (PGE2) transactivate EGFR through increased cell‐surface delivery of the EGFR ligand transforming growth factor‐α (TGFα) in polarizing madin‐darby canine kidney (MDCK) and Caco‐2 cells. This is achieved by PKA‐mediated phosphorylation of naked cuticle homolog 2 (NKD2), previously shown to bind TGFα and direct delivery of TGFα‐containing vesicles to the basolateral surface of polarized epithelial cells. VIP and PGE2 rapidly activate protein kinase A (PKA) that then phosphorylates NKD2 at Ser‐223, a process that is facilitated by the molecular scaffold A‐kinase anchoring protein 12 (AKAP12). This phosphorylation stabilized NKD2, ensuring efficient cell‐surface delivery of TGFα and increased EGFR activation. Thus, GPCR‐triggered, PKA/AKAP12/NKD2‐regulated targeting of TGFα to the cell surface represents a new mode of EGFR transactivation that occurs proximal to ligand cleavage by MMPs.

Integrin α5 clustering at the lateral membrane restores epithelial polarity in invasive colorectal cancer cells via deposition and polymerization of fibronectin and recruitment of paxillin to cluster sites, followed by tight junction formation., Apicobasolateral polarity is a fundamental property of epithelial cells, and its loss is a hallmark of cancer. Integrin-mediated contact with the extracellular matrix defines the basal surface, setting in motion E-cadherin–mediated cell–cell contact, which establishes apicobasolateral polarity. Role(s) for lateral integrins in this polarization process and the consequences of their disruption are incompletely understood. We show that addition of an integrin β1–activating monoclonal antibody, P4G11, to invasive colorectal cancer cells in three-dimensional type 1 collagen reverts the invasive phenotype and restores apicobasolateral polarity. P4G11 induces clustering of integrin α5β1 at lateral, intercellular surfaces. This leads to deposition and polymerization of fibronectin and recruitment of paxillin to sites of lateral integrin α5β1 clustering and is followed by tight junction formation, as determined by ZO-1 localization. Inducible elimination of integrin α5 abrogates the epithelial-organizing effects of P4G11. In addition, polymerization of fibronectin is required for the effects of P4G11, and addition of polymerized superfibronectin is sufficient to induce tight junction formation and apicobasolateral polarization. In the normal human colon, we show that integrin α5 localizes to the lateral membrane of terminally differentiated colonocytes and that integrin α5 staining may be reduced in colorectal cancer. Thus we propose a novel role for integrin α5β1 in regulating epithelial morphogenesis.

The anti-inflammatory and anti-tumor effects of berberine, a traditional Chinese medicine, were separately discovered in pathological intestinal tissues. However, whether the anti-inflammatory effect of berberine contributes to its anti-tumor effect on colitis-associated colorectal cancer (CACRC) remains unknown. In the present study, we found that berberine effectively inhibited colitis-associated tumorigenesis and colonic epithelium hyperproliferation in dextran sulfate sodium (DSS)-treated ApcMin/+ mice. A mechanistic study identified that these inhibitory effects of berberine occurred through blocking interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) expression in colonic macrophages. An in vitro study on cell lines identified that berberine treatment of Raw 264.7 macrophages resulted in conditioned media with fewer proliferative effects on a cell line with a heterozygous Apc mutation (Immorto-Min colonic epithelium, IMCE). EGFR-ERK signaling act downstream of berberine/pro-inflammatory cytokines axis to regulate CACRC cell proliferation. Furthermore, in vivo administration of IL-6 to DSS-treated ApcMin/+ mice effectively weakened the inhibitory effects of berberine on tumorigenesis and EGFR-ERK signaling in colon tissues. Altogether, the results of our studies have revealed that berberine inhibits the development of CACRC by interfering with inflammatory response-driven EGFR signaling in tumor cell growth. The findings of this study support the possibility that berberine and other anti-inflammatory drugs may be beneficial in the treatment of CACRC.

Cyto-nuclear shuttling of β-catenin is at the epicenter of the canonical Wnt pathway and mutations in genes that result in excessive nuclear accumulation of β-catenin are the driving force behind the initiation of many cancers. Recently, Naked Cuticle homolog 1 (Nkd1) has been identified as a Wnt-induced intracellular negative regulator of canonical Wnt signaling. The current model suggests that Nkd1 acts between Disheveled (Dvl) and β-catenin. Here, we employ the zebrafish embryo to characterize the cellular and biochemical role of Nkd1 in vivo. We demonstrate that Nkd1 binds to β-catenin and prevents its nuclear accumulation. We also show that this interaction is conserved in mammalian cultured cells. Further, we demonstrate that Nkd1 function is dependent on its interaction with the cell membrane. Given the conserved nature of Nkd1, our results shed light on the negative feedback regulation of Wnt signaling through the Nkd1-mediated negative control of nuclear accumulation of β-catenin.

In Drosophila, naked cuticle is an inducible antagonist of the Wnt-beta-catenin pathway, likely acting at the level of Dishevelled (Dsh/Dvl), an essential component of this pathway. The mechanism by which naked cuticle and its two vertebrate orthologs, Naked1 (NKD1) and Naked2 (NKD2), inhibit Dvl function is unknown. NKD2 is myristoylated, a co-translational modification that leads to its plasma membrane localization. In contrast, myristoylation-deficient G2A NKD2 is cytoplasmic. Herein we show that the ability of Nkd2/NKD2 to antagonize Wnt-beta-catenin activity during zebrafish embryonic development and in mammalian HEK293 cells is myristoylation-dependent. NKD2 and Dvl-1 interact and co-localize at the lateral membrane of polarized epithelial cells. In reciprocal overexpression and siRNA knockdown experiments, NKD2 and Dvl-1 destabilize each other via enhanced polyubiquitylation; this effect is also dependent upon Naked2 myristoylation. Cell fractionation and ubiquitylation assays indicate that endogenous NKD2 interacts with a slower migrating, ubiquitylated form of Dvl-1 in plasma membrane fractions. These results provide a mechanism by which NKD2 antagonizes Wnt signaling: myristoylated NKD2 interacts with Dvl-1 at the plasma membrane, and this interaction leads to their mutual ubiquitin-mediated proteasomal degradation.

Autosomal dominant (ADPKD) and autosomal recessive (ARPKD) polycystic kidney disease are caused by mutations in Pkd1/Pkd2 and Pkhd1, which encode polycystins (PCs) and fibrocystin/polyductin (FPC). Our recent study reported that a deficiency in FPC increases the severity of cystic disease in Pkd2 mutants and down-regulates PC2 in vivo, but the precise molecular mechanism of these effects is unknown (Kim, I., Fu, Y., Hui, K., Moeckel, G., Mai, W., Li, C., Liang, D., Zhao, P., Ma, J., Chen, X.-Z., George, A. L., Jr., Coffey, R. J., Feng, Z. P., and Wu, G. (2008) J. Am. Soc. Nephrol. 19, 455–468). In this study, through the use of deletion and mutagenesis strategies, we identified a PC2-binding domain in the intracellular C terminus of FPC and an FPC-binding domain in the intracellular N terminus of PC2. These binding domains provide a molecular basis for the physical interaction between PC2 and FPC. In addition, we also found that physical interaction between the binding domains of PC2 and FPC is able to prevent down-regulation of PC2 induced by loss of FPC. In vivo, we generated a mouse model of ADPKD with hypomorphic Pkd2 alleles (Pkd2nf3/nf3) and show that PC2 down-regulation is accompanied by a phenotype similar to that of Pkhd1–/– mice. These findings demonstrate a common mechanism underlying cystogenesis in ADPKD and ARPKD and provide insight into the molecular relationship between PC2 and FPC.

By interacting with the cytoplasmic tail of a Golgi-processed form of transforming growth factor-alpha (TGFalpha), Naked2 coats TGFalpha-containing exocytic vesicles and directs them to the basolateral corner of polarized epithelial cells where the vesicles dock and fuse in a Naked2 myristoylation-dependent manner. These TGFalpha-containing Naked2-associated vesicles are not directed to the subapical Sec6/8 exocyst complex as has been reported for other basolateral cargo, and thus they appear to represent a distinct set of basolaterally targeted vesicles. To identify constituents of these vesicles, we exploited our finding that myristoylation-deficient Naked2 G2A vesicles are unable to fuse at the plasma membrane. Isolation of a population of myristoylation-deficient, green fluorescent protein-tagged G2A Naked2-associated vesicles was achieved by biochemical enrichment followed by flow cytometric fluorescence-activated vesicle sorting. The protein content of these plasma membrane de-enriched, flow-sorted fluorescent G2A Naked2 vesicles was determined by LC/LC-MS/MS analysis. Three independent isolations were performed, and 389 proteins were found in all three sets of G2A Naked2 vesicles. Rab10 and myosin IIA were identified as core machinery, and Na(+)/K(+)-ATPase alpha1 was identified as an additional cargo within these vesicles. As an initial validation step, we confirmed their presence and that of three additional proteins tested (annexin A1, annexin A2, and IQGAP1) in wild-type Naked2 vesicles. To our knowledge, this is the first large scale protein characterization of a population of basolaterally targeted exocytic vesicles and supports the use of fluorescence-activated vesicle sorting as a useful tool for isolation of cellular organelles for comprehensive proteomics analysis.

Transforming growth factor-alpha (TGF-alpha) is the major autocrine EGF receptor ligand in vivo. In polarized epithelial cells, proTGF-alpha is synthesized and then delivered to the basolateral cell surface. We previously reported that Naked2 interacts with basolateral sorting determinants in the cytoplasmic tail of a Golgi-processed form of TGF-alpha and that TGF-alpha is not detected at the basolateral surface of Madin-Darby canine kidney (MDCK) cells expressing myristoylation-deficient (G2A) Naked2. By high-resolution microscopy, we now show that wild-type, but not G2A, Naked2-associated vesicles fuse at the plasma membrane. We further demonstrate that Naked2-associated vesicles are delivered to the lower lateral membrane of polarized MDCK cells independent of mu1B adaptin. We identify a basolateral targeting segment within Naked2; residues 1-173 redirect NHERF-1 from the apical cytoplasm to the basolateral membrane, and internal deletion of residues 37-104 results in apical mislocalization of Naked2 and TGF-alpha. Short hairpin RNA knockdown of Naked2 leads to a dramatic reduction in the 16-kDa cell surface isoform of TGF-alpha and increased cytosolic TGF-alpha immunoreactivity. We propose that Naked2 acts as a cargo recognition and targeting (CaRT) protein to ensure proper delivery, tethering, and fusion of TGF-alpha-containing vesicles to a distinct region at the basolateral surface of polarized epithelial cells.

Naked1 and 2 are two mammalian orthologs of Naked Cuticle, a canonical Wnt signaling antagonist in Drosophila. Naked2, but not Naked1, interacts with transforming growth factor-alpha (TGFalpha) and escorts TGFalpha-containing vesicles to the basolateral membrane of polarized epithelial cells. Full-length Naked2 is poorly soluble. Since most functional domains, including the Dishevelled binding region, EF-hand, vesicle recognition, and membrane targeting motifs, reside in the N-terminal half of the protein, we expressed and purified the first 217 residues of human Naked2 and performed a functional analysis of this fragment. Its circular dichroism (CD) and nuclear magnetic resonance (NMR) spectra showed no evidence of secondary and/or tertiary structure. The fragment did not bind calcium or zinc. These results indicate that the N-terminal half of Naked2 behaves as an intrinsically unstructured protein.

Cytokinesis and septation in the fission yeast Schizosaccharomyces pombe are studied as a model for mammalian cell division. In fission yeast, septation is positively regulated by Spg1, a Ras family GTPase that localizes to spindle-pole bodies (SPBs) throughout the cell cycle. As cells enter mitosis, Spg1 accumulates in an active, GTP-bound form and binds the Cdc7 protein kinase to cause Cdc7 translocation to SPBs. Cdc7 disappears from one SPB in mid-anaphase and from the second SPB in late mitosis. Byr4 plus Cdc16 negatively regulate septation by forming a two-component GTPase-activating protein for Spg1. These results led us to hypothesize that Byr4 localization to SPBs regulated the nucleotide state of Spg1, due to its ability to form Spg1GAP activity with Cdc16 and thus the binding of Cdc7 to Spg1 at SPBs. To test this hypothesis, Byr4 localization was determined using indirect immunofluorescence. This analysis revealed that Byr4 was localized to SPBs that did not contain Cdc7. In byr4(-) mutants, Cdc7 localized to interphase SPBs and only symmetrically localized to mitotic SPBs. In contrast, Byr4 overexpression prevented Spg1 and Cdc7 localization to SPBs. These results suggest that Byr4 localization to SPBs maintains Spg1 in an inactive form, presumably by stimulating Spg1 GTPase activity with Cdc16, and that loss of Byr4 from mitotic SPBs increases the active fraction of Spg1 and thereby increases Spg1-Cdc7 binding. Byr4 localization to SPBs was decreased in spg1, cdc16, sid4, and cdc11 mutants as well as in several mutants that affect medial F-actin structures, suggesting that multiple pathways regulate Byr4 localization to SPBs.